Chapter 19 Spectrophotometry : Instruments & Application
19.1 The Spectrophotometer –1 Remote sensing of airborne bacteria: Optical fiber coated with antibodies to detect spores of a specific bacterium
19.1 The Spectrophotometer -1 1)Spectrophotometer a) Single-beam b) Double-beam
19.1 The Spectrophotometer -2
19.1 The Spectrophotometer -3
19.1 The Spectrophotometer –4 1) Light source a.Tungsten lamp: Vis. near IR (320 nm~2500 nm) b.Deuterium are lamp: UV (200~400 nm) c.Electric discharge lamp + Hg (g) or Xenon: Vis & UV d.Globar (silicon carbide rod): IR (5000~200 cm -1 ) e.Laser: intense monochromatic sources.
19.1 The Spectrophotometer -5
19.1 The Spectrophotometer -6 2) Monochromator consists: (1) lenses or mirrors: focus the radiation (2) entrance and exit slits: restrict unwanted and control the spectral purity of radiation. (3) dispersing medium: separate the of polychromatic radiation from the source. (a) prism and (b) diffraction grating see Fig 19-2
19.1 The Spectrophotometer -7 a.entrance slit b.collimating mirror or lens c.a prism or grating d.focal plane e.exit slit 2) Monochromator
19.1 The Spectrophotometer -8 Interference of adjacent waves that are (a) 0°, (b) 90 °, and © 180 ° out of phase 2) Monochromator
19.1 The Spectrophotometer -9 nλ = a – b (n = 1 first-order….) Grating equation : nλ = d (sin – sin ) Filters: select a desired wavelength 2) Monochromator
19.1 The Spectrophotometer -10 Choosing the bandwidth: exit slit width Resolution Signal 2) Monochromator trade-off
19.1 The Spectrophotometer -10 3)Detector Convert radiant energy (photons) into an electrical signal Ideal detector : high sensitivity, high signal/noise, constant response for λs, and fast response time.
19.1 The Spectrophotometer -11 Detector response depends on the λ of the incident photons. 3) Detector
19.1 The Spectrophotometer -12 Photomultiplier tube: very sensitive detector
19.1 The Spectrophotometer -13 vs. Dispersive spectrophotometer: one at a time speed (~1s/spetrum) excellent λ repeatability measure λs simultaneously relatively insensitive to errors from stray light relatively poor resolution (1~3 nm) vs 0.1nm Photodiode array spectrophotometer : records the entire spectrum at once.
19.1 The Spectrophotometer -14 diode array spectrophotometer
19.2 Analysis of a mixture -1 1)Absorbance of a mixture : A = e x b[X] + e y b[Y] + …
19.2 Analysis of a mixture -2 2)Isosbestic points : for rxn: X Y, every spectrum recorded during chemical reaction will cross at the same point. Good evidence for only two principle species in rxn. Ex: HIn In - + H +
19.2 Analysis of a mixture -3 Why isosbestic point?
19.3 Spectrophotometric Titrations -1 apotransferrin + 2Fe 3+ (Fe 3+ ) 2 transferrin colorless red (465 nm)
Ferric nitrilotriacetate [used to avoid Fe(OH)3 ] 19.3 Spectrophotometric Titrations-2
Correcting A for the effect of dilution 125 μL ferric nitrilotriancetate 2 mL apotransferrin A = A corrected = ? 19.3 Spectrophotometric Titrations-3 ex.at p.408
19.4 What happens when a molecule absorbs light ? 1)Absorbing species : M + hν M* (lifetime : ~ sec) Relaxation processes : a)M* M + heat (most common) b)M* new species (photochemical reaction) c)M* M + h (fluorescence, phosphorescence)
19.4 Geometry of formaldehyde
2)Types of absorbing electrons Consider formaldehyde: three types of molecular orbitals 19.4 What happens when a molecule absorbs light ?
19.4 What happens absorbs light ? MO of CH 3 CO
Four types of electronic transitions σ*σ* π*π* σ π n E < 125 nm 150~250 nm 200~700 nm 19.4 What happens when a molecule absorbs light ?
3)Singlet / Triplet excited states E: T 1 < S 1 ground singlet state excited singlet (S 1 ) excited triplet (T 1 ) 19.4 What happens when a molecule absorbs light ? -5
4)Electronic transition of formaldehyde n * (T 1 ), absorption of light at λ = 397 nm green-yellow n * (S 1 ), absorption of light at λ = 355 nm colorless (more probable) 19.4 What happens when a molecule absorbs light ? -6
19.4 What happens when absorbs light ? 5)Vibrational & Rotational states of CH 3 CO (IR and microwave radiation)
6)What happens to absorbed energy 19.4 a molecule absorbs light
7) Luminescence procedures : emission spectrum of M* provides information for qualitative or quantitative analysis. Photoluminescence : a)Fluorescence : S 1 S 0, no change in electron spin. (< s) b)Phosphorescence : T 1 S 0, with a change in electron spin. (10 -4 ~10 2 s) Chemiluminescence : Chemical reaction (not initiated by light) release energy in the form of light. ex : firefly a molecule absorbs light
7)In which your class really shines ? emission spectrum 19.4 a molecule absorbs light
8)Absorption & Emission Spectra
19.5 Luminescence in analytical chemistry 1) Instrument .hν out (photon) heat breaking a chemical bond hν in
19.5 Luminescence 2) I = kP o C incident radiation sensitivity by P 0 or C 3)more sensitive than Absorption
4) Fluorimetric Assay of Selenium in Brazil Nuts –Se is a trace element essential to life: destruct ROOH (free radical) –Derivatized: –Self-absorption: quench 19.5 Luminescence
5) Immunoassarys employ anitbody to detect analyte. Ex: ELISA
19.5 Luminescence a.pregnancy test. sensitive to < 1 ng of analyte b.Enviromental Analysis. (ppm) or (ppt) pesticides, industrial chemicals, & microbialtoxins.
Environmental Analysis 19.5 Luminescence
19-14
19-15
19-18
19-21
19-22